Eric Bellefroid

X-MyT1, a Xenopus C2HC-type zinc finger protein with a regulatory function in neuronal differentiation.

X-MyT1 is a C2HC-type zinc finger protein that we find to be involved in the primary selection of neuronal precursor cells in Xenopus. Expression of this gene is positively regulated by the bHLH protein X-NGNR-1 and negatively regulated by the Notch/Delta signal transduction pathway. X-MyT1 is able to promote ectopic neuronal differentiation and to confer insensitivity to lateral inhibition, but only in cooperation with bHLH transcription factors. Inhibition of X-MyT1 function inhibits normal neurogenesis as well as ectopic neurogenesis caused by overexpression of X-NGNR-1. On the basis of these findings, we suggest that X-MyT1 is a novel, essential element in the cascade of events that allows cells to escape lateral inhibition and to enter the pathway that leads to terminal neuronal differentiation.

Clustered Organization of Homologous Krab Zinc-finger Genes With Enhanced Expression in Human T-lymphoid Cells

KRAB zinc‐finger proteins (KRAB‐ZFPs) constitute a large subfamily of ZFPs of the Krüppel C2H2 type. KRAB (Krüppel‐associated box) is an evolutionarily conserved protein domain found N‐terminally with respect to the finger repeats. We report here the characterization of a particular subgroup of highly related human KRAB‐ZFPs. ZNF91 is one representative of this subgroup and contains 35 contiguous finger repeats at its C‐terminus. Three mRNA isoforms with sequence identity to ZNF91 were isolated by the polymerase chain reaction. These encode proteins with a KRAB domain present, partially deleted or absent. Five genomic fragments were characterized, each encoding part of a gene: the ZNF91 gene or one of four distinct, related KRAB‐ZFP genes. All exhibit a common exon/intron organization with the variant zinc finger repeats organized in a single exon and the KRAB domain encoded by two separate exons. This positioning of introns supports the hypothesis that the mRNA isoforms encoding polypeptides with variability in the KRAB domain could arise by alternative splicing. By in situ chromosomal mapping studies and by analysis of fragments from a human genomic yeast artificial chromosome library containing KRAB‐ZFP genes, we show that these genes occur in clusters; in particular, a gene complex containing over 40 genes has been identified in chromosomal region 19p12‐p13.1. These ZNF91‐related genes probably arose late during evolution since no homologous genes are detected in the mouse and rat genomes. Although the transcription of members of this KRAB‐ZFP gene subgroup is detectable in all human tissues, their expression is significantly higher in human T lymphoid cells.

Perspectives on zinc finger protein function and evolution--an update.

Complexity is one of the hallmarks that applies to C2H2 type zinc finger proteins (ZFPs). Structurally distinct clusters of zinc finger modules define an extremely large superfamily of nucleic acid binding proteins with several hundred, perhaps thousands of different members in vertebrates. Recent discoveries have provided new insights into the biochemistry of RNA and DNA recognition, into ZFP evolution and genomic organization, and also into basic aspects of their biological function. However, as much as we have learned, other fundamental questions about ZFP function remain highly enigmatic. This essay is meant to define what we personally feel are important questions, rather than trying to provide a comprehensive, encyclopaedic review.

Evolutionarily conserved role of nucleostemin: controlling proliferation of stem/progenitor cells during early vertebrate development.

ABSTRACT Nucleostemin (NS) is a putative GTPase expressed preferentially in the nucleoli of neuronal and embryonic stem cells and several cancer cell lines. Transfection and knockdown studies indicated that NS controls the proliferation of these cells by interacting with the p53 tumor suppressor protein and regulating its activity. To assess the physiological role of NS in vivo, we generated a mutant mouse line with a specific gene trap event that inactivates the NS allele. The corresponding NS−/− embryos died around embryonic day 4. Analyses of NS mutant blastocysts indicated that NS is not required to maintain pluripotency, nucleolar integrity, or survival of the embryonic stem cells. However, the homozygous mutant blastocysts failed to enter S phase even in the absence of functional p53. Haploid insufficiency of NS in mouse embryonic fibroblasts leads to decreased cell proliferation. NS also functions in early amphibian development to control cell proliferation of neural progenitor cells. Our results show that NS has a unique ability, derived from an ancestral function, to control the proliferation rate of stem/progenitor cells in vivo independently of p53.

dapk1, encoding an activator of a p19ARF-p53-mediated apoptotic checkpoint, is a transcription target of p53.

The p53 tumour suppressor functions as a transcriptional activator, and several p53-inducible genes that play a critical proapoptotic role have been described. Moreover, p53 regulates the expression of various proteins participating in autoregulatory feedback loops, including proteins that negatively control p53 stability (Mdm2 and Pirh2) or modulate stress-induced phosphorylation of p53 on Ser-46 (p53DINP1 or Wip1), a key event for p53-induced apoptosis. Here, we describe a new systematic analysis of p53 targets using oligonucleotide chips, and report the identification of dapk1 as a novel p53 target. We demonstrate that dapk1 mRNA levels increase in a p53-dependent manner in various cellular settings. Both human and mouse dapk1 genomic loci contain DNA sequences that bind p53 in vitro and in vivo. Since dapk1 encodes a serine/threonine kinase previously shown to suppress oncogene-induced transformation by activating a p19ARF/p53-dependent apoptotic checkpoint, our results suggest that Dapk1 participates in a new positive feedback loop controlling p53 activation and apoptosis.

Multihormonal regulation of the human prolactin gene expression from 5000 bp of its upstream sequence

We have cloned DNA sequences extending up to 6000 bp upstream from the first exon of the human prolactin (hPRL) gene. 5000 bp of these upstream sequences were fused to a CAT reporter gene and shown to provide tissue-specific transient expression in rat pituitary GH3 cells. Multihormonal response was found in this transient expression assay, leading to significant 2- to 5-fold induction by addition of 8-chlorophenylthio-cyclic AMP, thyrotropin-releasing hormone, epidermal growth factor, basic fibroblast growth factor, phorbol myristate acetate, a calcium channel agonist (Bay K-8644) and triiodothyronine. A 3-fold inhibition was observed in the presence of the glucocorticoid agonist dexamethasone. The sequence of the hPRL promoter was determined up to coordinate -3470. Computer similarity search between the rat and human sequences showed two highly conserved regions corresponding to the proximal and distal tissue specific enhancers described in both PRL promoters.

Atypical Mowat–Wilson patient confirms the importance of the novel association between ZFHX1B/SIP1 and NuRD corepressor complex

Mutations in ZFHX1B cause Mowat-Wilson syndrome (MWS) but the precise mechanisms underlying the aberrant functions of mutant ZFHX1B proteins (also named Smad-interacting protein-1, SIP1) in patients are unknown. Using mass spectrometry analysis, we identified subunits of the NuRD corepressor complex in affinity-purified Zfhx1b complexes. We find that Zfhx1b associates with NuRD through its N-terminal domain, which contains a previously postulated NuRD interacting motif. Interestingly, this motif is substituted by an unrelated sequence in a recently described MWS patient. We show here that such aberrant ZFHX1B protein is unable to recruit NuRD subunits and displays reduced transcriptional repression activity on the XBMP4 gene promoter, a target of Zfhx1b. We further demonstrate that the NuRD component Mi-2beta is involved in repression of the Zfhx1b target gene E-cadherin as well as in Zfhx1b-induced neural induction in animal caps from Xenopus embryos. Thus, NuRD and Zfhx1b functionally interact, and defective NuRD recruitment by mutant human ZFHX1B can be a MWS-causing mechanism. This is the first study providing mechanistic insight into the aberrant function of a single domain of the multi-domain protein ZFHX1B/SIP1 in human disease.

deltaEF1 and SIP1 are differentially expressed and have overlapping activities during Xenopus embryogenesis.

The zinc finger/homeodomain transcription factor (zfhx1) family in vertebrates consists of two members, δEF1 and SIP1. They have been proposed to display antagonistic activities in the interpretation of Smad‐dependent TGFβ signaling during mesoderm formation. We cloned Xenopus δEF1 cDNA, analyzed the expression profile of the gene, and compared the inducing and interacting properties of the protein to that of XSIP1. Whereas XSIP1 RNA is selectively expressed in the early developing nervous system, we show that XδEF1 gene transcription is only activated during neurulation and that its expression is restricted to the paraxial mesoderm. From early tail bud stage, XδEF1 and XSIP1 are coexpressed in migratory cranial neural crest, in the retina, and in the neural tube. Overproduction of XδEF1 in RNA‐injected embryos, like that of XSIP1, reduced the expression of BMP‐dependent genes but only XSIP1 has the ability to induce neural markers. We find that XδEF1 and XSIP1 can both form complexes, although with different efficiency, with Smad3, with the coactivators p300 and pCAF, and with the corepressor CtBP1. Together, these results indicate that δEF1 and SIP1 do not function as antagonists during Xenopus early embryogenesis but do display different repression efficiencies and interaction properties. Developmental Dynamics 235:1491–1500, 2006.

Transcription Factor Zic2 Inhibits Wnt/β-Catenin Protein Signaling

The Zic transcription factors play critical roles during embryonic development. Mutations in the ZIC2 gene are associated with human holoprosencephaly, but the etiology is still unclear. Here, we report a novel function for ZIC2 as a regulator of β-catenin·TCF4-mediated transcription. We show that ZIC2 can bind directly to the DNA-binding high mobility group box of TCF4 via its zinc finger domain and inhibit the transcriptional activity of the β-catenin·TCF4 complex. However, the binding of TCF4 to DNA was not affected by ZIC2. Zic2 RNA injection completely inhibited β-catenin-induced axis duplication in Xenopus embryos and strongly blocked the ability of β-catenin to induce expression of known Wnt targets in animal caps. Moreover, Zic2 knockdown in transgenic Xenopus Wnt reporter embryos led to ectopic Wnt signaling activity mainly at the midbrain-hindbrain boundary. Together, our results demonstrate a previously unknown role for ZIC2 as a transcriptional regulator of the β-catenin·TCF4 complex.

A role for Xenopus Gli-type zinc finger proteins in the early embryonic patterning of mesoderm and neuroectoderm

Gli-type zinc finger proteins play important regulatory roles in vertebrate and invertebrate embryogenesis. In Xenopus, the Gli-type proteins XGli-3 and XGli-4 are first expressed in earliest stages of mesoderm and neural development. Transient transfection assays reveal that XGli-3 and XGli-4 can function as transcription repressors. Counteracting the Gli-protein repressor activity by ectopic expression of a fusion protein that contains the Gli-zinc finger cluster connected to the E1A activator domain in Xenopus embryos results in specific morphological alterations in the developing somites and in the central nervous system. Altered expression characteristics for a broad set of molecular markers highlighting specific aspects of mesodermal and neural differentiation demonstrate an important role for Gli-type zinc finger proteins in the early mesodermal and neural patterning of Xenopus embryos.